During the fiscal year 2013-2014, we extended our efforts to elucidate the DNA sequence patterns guiding rotational and translational positioning of nucleosomes. In particular, we developed a novel DNA threading algorithm correctly predicting positioning of nucleosomes precisely mapped both in vitro and in vivo (in collaboration with F. Cui, Rochester Inst. of Technology, NY). We also ran all-atom energy minimization of numerous double-stranded DNA fragments undergoing conformational transitions similar to those observed in crystallized nucleosomes. This combined approach allowed us to make an important step forward, toward understanding the nucleosome code encripted in genomic DNA. The folding of DNA in nucleosomes is accompanied by the lateral displacements of adjacent base pairs, which are usually ignored. We have found, however, that the shear deformation, called Slide, plays a much more important role in DNA folding than was previously imagined. First, the lateral Slide deformations observed at sites of local anisotropic bending of DNA define its superhelical trajectory in chromatin. Second, the computed cost of deforming DNA on the nucleosome is sequence-specific: in optimally positioned sequences the most easily deformed base-pair steps (CA:TG and TA) occur at the sites of large positive Slide and negative Roll (where the DNA strongly bends, or kinks, into the minor groove). Here, we incorporate all the degrees of freedom of 'real' DNA, thereby going beyond the limits of the conventional model ignoring the lateral Slide displacements of base pairs. Note that our results are in remarkable agreement with the in vitro sequence selection (SELEX) experiments. The successful prediction of nucleosome positioning for sequences of various GC-content demonstrates the potential advantage of our structural analysis, based on calculations of the DNA deformation energy. Recently, we developed a novel computational approach for calculation of topological polymorphism of the higher-order chromatin organization, the so-called 30-nm fibril. (This is important because the questions related to the structure of the 30-nm fibril still remain unanswered unambiguously. Most likely, the solenoid-type fibers proposed initially may be formed only for the inter-nucleosomal linkers L=50 bp or longer. When the linker L is 30 bp or shorter, which corresponds to chromatin in yeast and in human neurons, the two-start nucleosome fibers are formed.) We analyzed the two-start chromatin fibers for linkers L=13 to 37 bp. By optimizing the fiber energy with respect to the superhelical parameters we found two types of topological transition in fibers: one caused by an abrupt change in the linker DNA twisting, and another caused by over-crossing of the linkers. (The first transition is characterized by change in the DNA linking number, delta(Lk) = 1, and the second one by delta(Lk) = 2.) To the best of our knowledge, this topological polymorphism of the chromatin fibers was not reported in the computations published earlier. Importantly, the optimal configurations of the fibers with linkers L = 10n and 10n+5 bp are topologically different. Our results are consistent with experimental observations, such as the inclination 60-70 degrees (the angle between the nucleosomal disks and the fiber axis), helical rise, diameter and left-handedness of the fibers. In addition, we make several testable predictions, among them existence of different degree of DNA supercoiling in the fibers with L = 10n and 10n+5 bp, different stiffness of the two types of fibers, and a correlation between the local NRL and the level of transcription in different parts of the yeast genome. To test these predictions, we are planning two types of experiments. The first will be measuring the linking number difference in the plasmids containing arrays of '601' nucleosomes separated by linkers L = 20 and 25 bp (in collaboration with S. Grigoryev, Penn State). We will also analyze rearrangement of nucleosome positioning upon activation of transcription in yeast. Using the combined MNase/exoIII digestion described above will be essential for mapping nucleosomes with the highest possible resolution.